Highly productive alpha-amylases

ABSTRACT

The invention relates to mutant α-amylases that may be produced at high yield from recombinant microorganisms.

TECHNICAL FIELD

The present invention relates to mutant α-amylases having improvedproductivity.

BACKGROUND ART

α-Amylases [EC.3.2.1.1.] have been used in a wide range of industrialfields such as starch industry, brewing industry, fiber industry,pharmaceutical industry and food industry. Among them, those capable ofdegrading starches at high random are suited for detergents.Conventionally known as such are, as well as α-amylases derived fromBacillus licheniformis, liquefying alkaline α-amylases derived from thealkaliphilic strain Bacillus sp. KSM-AP1378 (FERM BP-3048) (WO94/26881)and improved enzymes having improved heat resistance and oxidantresistance (WO98/44126).

The present inventors have recently found liquefying alkaline α-amylasesderived from the alkaliphilic strain Bacillus sp. KSM-K38 (FERM BP-6946)and having chelating-agent- and oxidation-resistance (Japanese PatentApplication No. Hei 10-362487, Japanese Patent Application No. Hei10-362488); and improved enzymes having improved heat resistance(Japanese Patent Application No. Hei 11-163569).

In addition to such properties, enzymes for detergents are required tohave high productivity in consideration of their industrial production.Although various trials have been made to improve the heat resistance oroxidant resistance of α-amylases for detergent by using proteinengineering technique, neither improvement of productivity has beenconsidered sufficiently nor an attempt of production increase bymutation of a structural gene has been reported yet.

An object of the present invention is to provide mutant α-amylaseshaving excellent productivity.

DISCLOSURE OF THE INVENTION

The present inventors introduced, in microorganisms, mutant α-amylasestructural gene constructed by site-directed mutagenesis and evaluatedproductivity of α-amylases. As a result, it has been found that since an(α-amylase gene has a site taking part in the improvement ofproductivity, introduction, into a microorganism, of a recombinant genehaving this site mutated makes it possible to produce α-amylases havingdrastically improved productivity.

In one aspect of the present invention, there is thus provided a mutantα-amylase which is derived from an α-amylase having an amino acidsequence represented by SEQ ID No. 1 or showing at least 60% homologythereto by substitution or deletion of at least one amino acid residuecorresponding to any one of Pro₁₉, Gln₈₆, Glu₁₃₀, Asn₁₅₄, Arg₁₇₁,Ala₁₈₆, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, Glu₂₇₆, Asn₂₇₇, Arg₃₁₀,Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁ and Gly₄₇₆ of the amino acidsequence.

In another aspect of the present invention, there is also provided amutant α-amylase derived from an α-amylase having an amino acid sequencerepresented by SEQ ID No. 2 or showing at least 60% homology thereto bysubstitution or deletion of at least one amino acid residuecorresponding to any one of Asp₁₂₈, Gly₁₄₀, Ser₁₄₄, Arg₁₆₈, Asn₁₈₁,Glu₂₀₇, Phe₂₇₂, Ser₃₇₅, Trp₄₃₄ and Glu₄₆₆ of the amino acid sequence.

In a further aspect of the present invention, there is also provided agene encoding this mutant α-amylase, a vector containing the gene, acell transformed with the vector and a production method of a mutantα-amylase which comprises cultivating the transformed cell.

In a still further aspect of the present invention, there is alsoprovided a detergent composition containing this mutant α-amylase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of constructing a recombinant plasmid forproduction of an α-amylase derived from the strain KSM-1378 or KSM-K38.

FIG. 2 is a schematic view illustrating a method of introducing amutation into an α-amylase gene derived from the strain KSM-1378 orKSM-K38.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “highly productive mutant α-amylase” as used herein means anα-amylase whose yield is increased, upon production of it by cultivatinga recombinant microorganism, by at least 5%, preferably at least 10%,more preferably at least 20% compared with that before mutation.

The mutant α-amylase of the present invention is constructed so that outof amino acids constituting the α-amylase, the amino acid residuestaking part in the productivity are substituted with another amino acidresidues or deleted. Examples of the α-amylase usable here includeliquefying α-amylases derived from Bacillus. amyloliquefaciens orBacillus. licheniformis and liquefying alkaline α-amylases derived fromalkaliphilic microorganisms belonging to the Bacillus sp., of whichα-anylases having an amino acid sequence represented by SEQ ID No. 1 orSEQ ID No. 2 and α-amylases having at least 60% homology to theabove-described amino acid sequence are preferred.

Examples of the α-amylase having the amino acid sequence represented bySEQ ID No. 1 or α-amylase having at least 60% homology thereto includeliquefying alkaline α-amylases derived from the strain Bacillus sp.KSM-AP1378 (FERM BP-3048) (Japanese Patent Application Laid-Open No. Hei8-336392) and improved enzymes of the above-described one in heatresistance and oxidant resistance which are constructed by proteinengineering technique (WO98/44126).

Examples of the α-amylase having the amino acid sequence represented bySEQ ID No. 2 or having at least 60% is homology thereto includeliquefying alkaline α-amylases derived from the strain Bacillus sp.KSM-K38 (FERM BP-6946) and improved enzymes of the above-described onein heat resistance which are constructed by protein engineeringtechnique (Japanese Patent Application No. Hei 11-163569).

The homology of an amino acid sequence is calculated by Lipman-Pearsonmethod (Science, 227, 1435(1985)).

The mutant α-amylase of the present invention can be obtained first bycloning, from a microorganism producing an α-amylase, a gene encodingthe α-amylase. For this purpose, ordinarily employed gene recombinanttechnique, for example, the method as described in Japanese PatentApplication Laid-Open No. Hei 8-336392 may be employed. Examples of thegene usable here include that represented by SEQ ID No. 3 or SEQ ID No.4 which encodes the amino acid sequence represented by SEQ ID No. 1 orSEQ ID No. 2. Mutant genes derived from the above-described ones andhaving improved heat resistance and oxidant resistance are also usable.

For mutation of the gene thus obtained by cloning, any site-directedmutagenesis ordinarily employed can be adopted. For example, mutationcan be conducted using a “Site-Directed Mutagenesis System Mutan-SuperExpress Km” kit (product of Takara Shuzo Co., Ltd.).

Mutation for obtaining highly productive α-amylases of the invention canbe attained, for example, by substitution or deletion, in an α-amylasehaving an amino acid sequence represented by SEQ ID No. 1 or having atleast 60% homology thereto, of at least one amino acid residuecorresponding to any one of Pro₁₈, Gln₈₆, Glu₁₃₀, Asn₁₅₄, Arg₁₇₁,Ala₁₈₆, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, Glu₂₇₆, Asn₂₇₇, Arg₃₁₀,Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁ and Gly₄₇₆ of the amino acidsequence; or by substitution or deletion, in another α-amylase having anamino acid sequence represented by SEQ ID No. 2 or having at least 60%homology thereto, of at least one amino acid residue corresponding toany one of AsP₁₂₈, Gly₁₄₀, Ser₁₄₄, Arg₁₆₈, Asn₁₈₁, Glu₂₀₇, Phe₂₇₂,Ser₃₇₅, Trp₄₃₄ and Glu₄₆₆ of the amino acid sequence. Preferredmutations include, in the amino acid sequence of SEQ ID No. 1,substitution of the amino acid residue corresponding to Pro₁₈ with Seror Thr, the amino acid residue corresponding to Gln₈₆ with Glu, theamino acid residue corresponding to Glu₁₃₀ with Val or Gln, the aminoacid residue corresponding to Asn₁₅₄ with Asp, the amino acid residuecorresponding to Arg₁₇₁ with Cys or Gln, the amino acid residuecorresponding to Ala₁₈₆ with Val or Asn, the amino acid residuecorresponding to Glu₂₁₂ with Asp, the amino acid residue correspondingto Val₂₂₂ with Glu, the amino acid residue corresponding to Tyr₂₄₃ withCys or Ser, the amino acid residue corresponding to Pro₂₆₀ with Glu, theamino acid residue corresponding to Lys₂₆₉ with Gln, the amino acidresidue corresponding to Glu₂₇₆ with His, the amino acid residuecorresponding to Asn₂₇₇ with Ser or Phe, the amino acid residuecorresponding to Arg₃₁₀ with Ala, the amino acid residue correspondingto Glu₃₆₀ with Gln, the amino acid residue corresponding to Gln₃₉₁ withGlu, the amino acid residue corresponding to Trp₄₃₉ with Arg, the aminoacid residue corresponding to Lys₄₄₄ with Arg, the amino acid residuecorresponding to Asn₄₇₁ with Asp or Glu, or the amino acid residuecorresponding to Gly₄₇₆ with Asp;

or substitution, in the amino acid sequence of SEQ ID No. 2, of theamino acid residue corresponding to ASP128 with Val or Gln, the aminoacid residue corresponding to Gly₁₄₀ with Ser, the amino acid residuecorresponding to Ser₁₄₄ with Pro, the amino acid residue correspondingto Arg₁₆₈ with Gln, the amino acid residue corresponding to Gln₁₈₁ withVal, the amino acid residue corresponding to Glu₂₇₀ with Asp, the aminoacid residue corresponding to Phe₂₇₂ with Ser, the amino acid residuecorresponding to Ser₃₇₅ with Pro, the amino acid residue correspondingto Trp₄₃₄ with Arg or the amino acid residue corresponding to Glu₄₆₆with Asp.

Among the mutations of the amino acid sequence of SEQ ID No. 1, those bysubstitution of the amino acid residue corresponding to Gln86 with Glu,the amino acid residue corresponding to Glu₁₃₀ with Val or Gln, theamino acid residue corresponding to Ala₁₈₆ with Asn, the amino acidresidue corresponding to Tyr₂₄₃ with Ser, the amino acid residuecorresponding to Pro₂₆₀ with Glu, the amino acid residue correspondingto Lys₂₆₉ with Gln, the amino acid residue corresponding to Asn₂₇₇ withPhe and the amino acid residue corresponding to Asn₄₇₁ with Asp or Glucan bring about improvement in solubility of the α-amylase in a culturemedium or desalted and concentrated solution thereof. More specifically,the above-described mutations make it possible to improve the residualactivity of the α-amylase in the supernatant after storage at 4° C. forone week in a desalted and concentrated solution by at least 5%,especially 10% compared with the activity before mutation. Accordingly,in the case of the mutant α-amylases of the present invention obtainedby such amino acid mutations, a fermented concentrate solution of a highconcentration is available at a high yield and enzyme formulationtreatment such as ultrafiltration after fermentation production can beconducted efficiently.

A combination of two or more substitutions or deletions of various aminoacid residues is also effective for such amino acid mutations. It isalso possible to use the above-exemplified mutation in combination witha mutation for improving enzymatic properties, for example, in anα-amylase having an amino acid sequence represented by SEQ ID No. 1 orhaving at least 60% homology thereto, a mutation for improving heatresistance by deleting amino acid residues corresponding to Arg₁₈₁, andGly₁₈₂, a mutation for improving oxidant resistance by substituting theamino acid residue corresponding to Met₂₂₂ with Thr or a mutation forimproving solubility by substituting the amino acid residuecorresponding Lys₄₈₄ with Gln; or in an α-amylase having an amino acidsequence represented by SEQ ID No. 2 or having at least 60% homologythereto, a mutation for further reinforcing oxidant resistance bysubstituting the amino acid residue corresponding to Met₁₀₇ with Leu ora mutation for heightening detergency of a laundry detergent bysubstituting the amino acid residue corresponding Glu₁₈₈ with Ile.

A mutant α-amylase is available at a high yield by appropriatelycombining a mutant α-amylase structural gene with a control gene and aproper plasmid vector, thereby constructing a plasmid for the productionof the α-amylase, introducing the resulting plasmid into a microorganismsuch as Bacillus subtilis or Escherichia coli, preferably, Bacillussubtilis and cultivating the resulting recombinant microorganism.

The mutant α-amylase thus obtained has improved productivity by about 10to 500% as shown later in Examples while maintaining biochemicalproperties as an enzyme, thus having excellent properties. By theabove-described mutation of the amino acid residues of liquefyingalkaline α-amylases having heat resistance, chelating agent resistance,oxidant resistance and high solubility, it is therefore possible tocreate useful enzymes having drastically improved productivity in arecombinant microorganism without losing the above-described originalproperties.

The detergent compositions of the present invention may contain, inaddition to the α-amylase of the invention, one or more than one enzymesselected from debranching enzymes (such as pullulanase, isoamylase andneopullulanase), α-glucosidase, glucoamylase, protease, cellulase,lypase, pectinase, protopectinase, pectate lyase, peroxidase, laccaseand catalase.

The detergent composition may contain, in addition, componentsordinarily added to a detergent, for example, surfactants such asanionic surfactants, amphoteric surfactants, nonionic surfactants andcationic surfactants, chelating agents, alkali agents, inorganic salts,anti-redeposition agents, chlorine scavengers, reducing agents,bleaching agents, fluorescent dye solubilizing agents, perfumes,anti-caking agents, enzyme activating agents, antioxidants, antiseptics,blueing agents, bleach activating agents, enzyme stabilizing agents andregulator.

The detergent compositions of the invention can be produced in a mannerknown per se in the art from a combination of the highly productiveα-amylase available by the above-described method and theabove-described known detergent components. The form of the detergentcan be selected according to the using purpose and examples includeliquid, powder and granule. The detergent compositions of the presentinvention are suited as laundry detergents, bleaching detergents,detergents for automatic dish washer, pipe cleaners, and artificialtooth cleaners, of which they are especially suited as laundrydetergents, bleaching detergents and detergents for automatic dishwasher.

The highly productive mutant α-amylases of the invention are also usableas starch liquefying saccharifying compositions. Moreover, these mutantα-amylases, after addition thereto of one or more than one enzymesselected from glucoamylase, maltase, pullulanase, isoamylase andneopullulanase, can be allowed to act on starches.

Furthermore, the mutant α-amylases of the present invention are usableas a desizing composition of fibers and an enzyme such as pullulanase,isoamylase or neopullulanase can be incorporated in the composition.

EXAMPLES

Measurement of amylase activity and protein content Amylase activity andprotein content of the enzymes each produced from recombinant Bacillussubtilis were measured in accordance with the below-described methods.

Amylase activity was measured by the 3,5-dinitrosalicylic acid method(DNS method). After reaction at 50° C. for 15 minutes in a reactionmixture of a 40 mM glycine-sodium hydroxide buffer (pH 10) containingsoluble starch, the reducing sugar thus formed was quantitativelyanalyzed by the DNS method. As the titer of the enzyme, the amount ofthe enzyme which formed reducing sugar equivalent to 1 μmol of glucosein one minute was defined as one unit.

The protein content was determined by “Protein Assay Kit” (product ofBio-Rad Laboratories) using bovine serum albumin as standard.

Referential Example 1 Screening of Liquefying Alkaline Amylase

About 0.5 g of soil was suspended in sterilized water and the resultingsuspension was heat treated at 80° C. for 15 minutes. The supernatant ofthe heat treated mixture was diluted with an adequate amount ofsterilized water, followed by applying to an isolating agar medium(Medium A). The medium was then cultured at 30° C. for 2 days to growcolonies. The colonies which formed transparent zones in theirperipheries due to starch dissolution were selected and isolated asamylase producing strains. The resulting isolated strains wereinoculated in Medium B, followed by aerobic shaken culture at 30° C. for2 days. After cultivation, the chelating agent (EDTA) resisting capacityof the supernatant obtained by centrifugation was measured and inaddition, the optimum working pH was measured. Thus, strain Bacillus sp.KSM-K38 (FERM BP-6946) was obtained. Medium A: Tryptone 1.5% Soytone0.5% Sodium chloride 0.5% Colored starch 0.5% Agar 1.5% Na₂Co₃ 0.5% (pH10) Medium B: Tryptone 1.5% Soytone 0.5% Sodium chloride 0.5% Solublestarch 1.0% Na₂CO₃ 0.5% (pH 10)

The mycological properties of strain KSM-K38 are shown in Table 1. TABLE1 Strain KSM-K38 (a) Observation under microscope Cells are rods of asize of 1.0 to 1.2 μm × 2.4 to 5.4 μm in the strain K36 and 1.0 to 1.2μm × 1.8 to 3.8 μm in the strain K38, and form an elliptical endospore(1.0 to 1.2 μm × 1.2 to 1.4 μm) at their subterminals or center. Theyhave flagella and are motile. Gram's staining is positive. Acidfastness: negative. (b) Growth in various culture mediums. The strainsare alikaliphilic so that 0.5% sodium carbonate was added to the culturemedium in the tests described hereinafter. Nutrient agar plate cultureGrowth of cells is good. Colony has a circular shape, with its surfacebeing smooth and its peripheral end being smooth. The color of thecolony is yellowish brown. Nutrient agar slant culture Cells can grow.Nutrient broth Cells can grow. Stab culture in nutrient-broth gelatinGrowth of cells is good. Liquefaction of gelatin is not observed. Litmusmilk medium No change in growth. (c) Physiological properties Nitratereduction and denitrification Nitrate reduction: positiveDenitrification: negative MR test Indeterminable because the medium isan alkaline medium. V-P test Negative Production of indole NegativeProduction of hydrogen sulfide Negative Hydrolysis of starch PositiveUtilization of citric acid Positive in Christensen's medium but negativein Koser's medium and Simmon's medium. Utilization of inorganic nitrogensources Nitrate is utilized but ammonium salts are not. Production ofcolorants Negative Urease Negative Oxidase Negative Catalase PositiveGrowth range Growth temperature range: 15 to 40° C., optimum growthtemperature: 30° C., growth pH range: pH 9.0 to 11.0, optimum growth pHrange: same Behavior on oxygen Aerobic O-F test Cells do not grow Sugarutilization L-galactose, D-xylose, L-arabinose, lactose, glycerin,melibiose, ribose, D-glucose, D-mannose, maltose, sucrose, trehalose,D-mannitol, starch, raffinose and D-fructose are utilized. Growth in asalt-containing medium Cells can grow when salt concentration is 12%,but not when salt concentration is 15%.

Referential Example 2 Cultivation of Strain KSM-K38

In the liquid medium B of Referential Example 1, the strain KSM-K38 wasinoculated, followed by aerobic shaken culture at 30° C. for 2 days. Theamylase activity (at pH 8.5) of each of the supernatants isolated bycentrifugation was measured. As a result, the activity in 1 L of theculture medium was found to be 1177 U.

Referential Example 3 Purification of Liquefying Alkaline Amylase

Ammonium sulfate was added to the supernatant of the culture medium ofthe strain KSM-K38 obtained in Referential Example 2 to give 80%saturation, followed by stirring. The precipitate thus formed wascollected and dissolved in a 10 mM tris-HCl buffer (pH 7.5) containing 2mM CaCl₂ to dialyze the resulting solution against the buffer overnight.The dialysate was loaded on a DEAE-Toyopearl 650M column equilibratedwith the same buffer and protein was eluted in a linear gradient of 0 to1 M of NaCl in the same buffer. The active fraction obtained by gelfiltration column chromatography after dialysis against the same bufferwas dialyzed against the buffer, whereby purified enzyme exhibited asingle band on polyacrylamide gel electrophoresis (gel concentration:10%) and sodium dodecylsulfate (SDS) electrophoresis was obtained.

Referential Example 4 Enzymological Properties

The properties of the purified enzyme are as follows:

(1) Action

It acts on starch, amylose, amylopectin and α-1,4-glycoside bond whichis a partially degraded product thereof to degrade them and produce,from amylose, glucose (G1), maltose (G2), maltotriose (G3),maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) andmaltoheptaose (G7). But it does not act on pullulan.

(2) pH Stability (Britton-Robinson Buffer)

It exhibits residual activity of 70% or more within a range of pH 6.5 to11.0 under treating conditions at 40° C. for 30 minutes.

(3) Working Temperature Range and Optimum Working Temperature

It acts in a wide temperature range of from 20 to 80° C., with theoptimum working temperature being 50 to 60° C.

(4) Temperature Stability

The temperature at which the enzyme loses its activity was examined bycausing a temperature change in a 50 mM glycine-sodium hydroxide buffer(pH 10) and then, treating at each temperature for 30 minutes. Theresidual activity of the enzyme is 80% or more at 40° C. and about 60%even at 45° C.

(5) Molecular Weight

The molecular weight as measured by sodium-dodecylsulfate polyacrylamidegel electrophoresis is 55,000±5,000.

(6) Isoelectric Point

Its isoelectric point as measured by isoelectric focusingelectrophoresis is about 4.2.

(7) Effects of Surfactants

It is almost free from activity inhibition (activity remaining ratio:90% or more) even when treated at pH 10 and 30° C. for 30 minutes in a0.1% solution of a surfactant such as sodium linear alkylbenzenesulfonate, alkyl sulfate ester sodium salt, polyoxyethylene alkylsulfateester sodium salt, sodium α-olefin sulfonate, sodium α-sulfonated fattyacid ester, sodium alkylsulfonate, SDS, soap and softanol.

(8) Effects of Metal Salts

It was treated at pH 10 and 30° C. for 30 minutes in each of thereaction systems containing various metal salts and their effects werestudied. Its activity is inhibited by 1 mM of Mn²⁺ (inhibition ratio:about 75%) and slightly inhibited by 1 mM of Sr²⁺ and Cd²⁺ (inhibitionratio: about 30%).

Example 1 Preparation of Various Recombinant Plasmids Having anα-Amylase Gene Ligated Thereto

In accordance with the method as described in WO98/44126, genes encodinga mutant α-amylase (which will hereinafter be described as “ΔRG”) havingimproved heat resistance and a mutant α-amylase (“ΔRG-M202T”) havingimproved oxidant resistance as well as improved heat resistance wereconstructed, respectively, by deleting Arg₁₈₁ and Gly₁₈₂ of theα-amylase (“LAMY”) which was derived from the strain Bacillus sp.KSM-AP1378 (FERM BP-3048) and had the amino acid sequence represented bySEQ ID No. 1; and by, in addition to this mutation by deletion,substituting Thr for Met₂₀₂ of the amino acid sequence represented bySEQ ID No. 1. With the genes as a template, gene fragments (about 1.5kb) encoding these mutant α-amylases were amplified by the PCR reactionusing primers LAUS (SEQ ID No. 5) and LADH (SEQ ID No. 6). After cuttingof them with a restriction enzyme SalI, each of the fragments wasinserted into the SalI-SmaI site of an expression vector pHSP64(Japanese Patent Application Laid-Open No. Hei 6-217781), whereby arecombinant plasmid having a structural gene of each of the mutantα-amylases bonded thereto was constructed downstream of a strongpromoter derived from an alkaline cellulase gene of the strain Bacillussp. KSM-64 (FERM P-10482).

In the meantime, with a chromosomal DNA, which had been extracted fromthe cells of the strain Bacillus sp. KSM-K38 (FERM BP-6946) by themethod of Saito and Miura (Biochim. Biophys. Acta, 72, 619(1961)), as atemplate, PCR reaction was effected using primers K38US (SEQ ID No. 7)and K38DH (SEQ ID No. 8) shown in Table 2, whereby a structural genefragment (about 1.5 kb) encoding an α-amylase (which will hereinafter bedescribed as “K38AMY”) having an amino acid sequence of SEQ ID No. 2 wasamplified. After cutting of it with a restriction enzyme SalI, theresulting fragment was inserted into the SalI-SmaI site of an expressionvector pHSP64 to construct, downstream of a strong promoter derived froman alkaline cellulase gene of the strain Bacillus sp. KSM-64 (FERMP-10482) contained in pHSP64, a recombinant plasmid having a structuralgene of K38AMY bonded thereto (FIG. 1). With this recombinant plasmid asa template, PCR reaction was effected using the primers CLUBG (SEQ ID.No. 9) and K38DH (SEQ. ID. 8) to amplify a gene fragment (about 2.1 kb)having the strong promoter and K38AMY bonded thereto.

By the recombinant PCR method as described below, a gene encodingchimeric α-amylase between K38AMY and LAMY was constructed. Describedspecifically, with a chromosomal DNA of the strain KSM-K38 (FERM BP6946)as a template, PCR reaction was conducted using primers K38DH (SEQ IDNo. 8) and LA-K38 (SEQ ID No. 10) shown in Table 2, whereby a fragmentencoding the sequence from Gln₂₀ downstream to the C-terminal of theamino acid sequence of K38AMY represented by SEQ ID No. 2 was amplified.With the above-described recombinant plasmid containing the LAMY geneand strong promoter as a template, PCR reaction was conducted usingprimers CLUBG (SEQ ID No. 9) and LA-K38R (SEQ ID No. 11) shown in Table2, whereby a gene fragment encoding from the upstream strong promoter toGly₂₁ of the amino acid sequence of LAMY of SEQ ID No. 1 was amplified.By the second PCR reaction using the resulting two DNA fragments andprimers CLUBG (SEQ ID No. 9) and K38DH (SEQ ID No. 8) shown in Table 2,the resulting two fragments having, at the end thereof, complementarysequences derived from primers LA-K38 (SEQ ID No. 10) and LA-K38R (SEQID No. 11) respectively were combined, whereby a gene fragment (about2.1 kb) encoding a chimeric α-amylase (which will hereinafter bedescribed as “LA-K38AMY”) which has, successively bonded thereto, aregion encoding from His₁ to Gly₂₁ of the LAMY downstream of the strongpromoter and a region encoding from Gln₂₀ to the C-terminal of theK38AMY was amplified.

By using a “Site-Directed Mutagenesis System Mutan-Super Express Km”kit(product of Takara Shuzo Co., Ltd.), the below-described mutations wereintroduced to the K38AMY and LA-K38AMY. First, the K38AMY and LA-K3.8AMYgene fragments (about 2.1 kb) were inserted into the site SmaI of aplasmid vector pKF19k attached to the kit to construct a mutagenicrecombinant plasmid (FIG. 2). A site-directed mutagenic oligonucleotideprimer N19OF (SEQ ID No. 50) shown in Table 2 was 5′-phosphorylated withT4 DNA kinase. Using this and the above-described mutagenic recombinantplasmid, mutagenesis was effected in accordance with the method of thekit and by using the reaction product, the strain Escherichia coliMV1184 (“Competent cell MV1184”, product of Takara Shuzo Co., Ltd.) wastransformed. From the transformant thus obtained, a recombinant plasmidwas extracted, followed by analysis of a basic sequence, wherebymutation by substitution of Phe for Asn₁₉₀ was confirmed. By repeatedintroduction of mutagenic reactions into the mutated gene bysuccessively using primers A209V (SEQ ID No. 51) and QEYK (SEQ ID No.49) in a similar manner as above, thereby substituting Asn₁₉₀ andGln₂₀₉, each of the amino acid sequence of the K38AMY represented by SEQID No. 2, with Phe and Val, respectively, and the sequence from Asp₁ toGly₁₉ of the amino acid sequence of the K38AMY represented by SEQ ID No.2 with the sequence from His₁ to Gly₂₁ of the amino acid sequence of theLAMY represented by SEQ ID NO. 1; by substituting Gln₁₆₇, Tyr₁₆₉, Asn₁₉₀and Gln₂₀₉, each of the amino acid sequence of the K38AMY, with Glu,Lys, Phe and Val, respectively and the sequence from Asp₁ to Gly₁₉ ofthe amino acid sequence of the K38AMY with the sequence from His₁ toGly₂₁ of the amino acid sequence of the LAMY; and substituting Gln₁₆₇and Tyr₁₆₉, Asn₁₉₀ and Gln₂₀₉, each of the amino acid sequence of theK38AMY, with Glu, Lys, Phe and Val, respectively, genes encoding amutant α-amylase (which will hereinafter be described as“LA-K38AMY/NFQV”) having improved heat resistance, a mutant α-amylase(“LA-K38AMY/QEYK/NFQV”) having drastically improved heat resistance, anda mutant α-amylase (“QEYK/NFQV”) having improved heat resistance wereconstructed, respectively.

With these genes as a template, PCR reaction was conducted using primersK38US (SEQ ID No. 7) and K38DH (SEQ ID No. 8) to amplify structural genefragments (about 1.5 kb) encoding the mutant α-amylases were amplified.They were then inserted into the SalI-SmaI site of an expression vectorpHSP64 in a similar manner as above, whereby a recombinant plasmidhaving structural genes of these mutant α-amylases bonded each other wasconstructed (FIG. 1).

Example 2 Introduction of a Mutation for Improving α-AmylaseProductivity

A “Site-Directed Mutagenesis System Mutan-Super Express Km” kit ofTakara Shuzo Co., Ltd. was used for site-directed mutagenesis forimproving amylase productivity of recombinant Bacillus subtilis. Withvarious recombinant plasmids obtained in Example 1 as a template, PCRreactions were effected using primers CLUBG (SEQ ID No. 9) and LADH (SEQID No. 6) for ΔRG and ΔRG/M202T, while using primers CLUBG (SEQ ID No.9) and K38DH (SEQ ID No. 8) for K38AMY, LA-K38AMY/NFQV,LA-K38AMY/QEYK/NFQV and QEYK/NFQV, whereby fragments of about 2.1 kbfrom the upstream strong promoter derived from the strain KSM-64 to thedownstream α-amylase gene were amplified. These amplified fragments wereinserted into the SmaI site of a plasmid vector pKF19k attached to theabove-described kit, whereby various mutagenetic recombinant plasmidswere constructed (FIG. 2).

Various oligonucleotide primers for site-directed mutagenesis shown inTable 2 (SEQ ID Nos. 12 to 51) were 5′-phosphorylated with T4DNA kinase,and by using the resultant products and the above mutageneticrecombinant plasmids, mutagenesis was conducted in accordance with themethod as described in the kit. With the reaction products, the strainEscherichia coli MV1184(“Competent Cell MV1184” product of Takara ShuzoCo., Ltd.) was transformed. From the resulting transformants, arecombinant plasmid was extracted, followed by analysis of a basesequence to confirm mutation. TABLE 2 SeQ ID Using No. Primer Basesequence (5′-3′) purpose 5 LAUS GAGTCGACCAGCACAAGCCCATCATAATGG PCR for 6LADH TAAAGCTTCAATTTATATTGG recombi- 7 K38USGGGTCGACCAGCACAAGCCGATGGATTGAACGGTACGATG nation 8 K38DHTAAAGCTTTTGTTATTGGTTCACGTACAC 9 CLUBG CCAGATCTACTTACCATTTTAGAGTCA 10LA-K38 ATTTGCCAAATGACGGGCAGCATTGGAATCGGTT 11 LA-K38RAACCGATTCCAATGCTGCCCGTCATTTGGCAAAT 12 P18STTTGAATGGCATTTGTCAAATGACGGGGAACCAC Site-directed 13 Q86EACAAGGAGTCAGTTGGAAGGTGCCGTGACATCT mutagenesis 14 E130VCGAAACCAAGTAATATCAGGT (ΔRG) 15 N154D AATACCCATTCCGATTTTAAATGGCGC 16R171C GATTGGGATCAGTCATGYCAGCTTCAGAACAAA 17 A186VAAATTCACCGGAAAGGTATGGGACTGGGAAGTA 18 E212D TCATCCAGATGTAATCAATG 19 V222ECTTAGAAATTGGGGAGAATGGTATACAAATACA 20 Y243CGTGAAACATATTAAATGCAGCTATACGAGAGAT 21 P260EAACACCACAGGTAAAGAAATGTTTGCAGTTGCA 22 K269Q AGAATTTTGGCAAAATGACCT 23E276H TTGCTGCAATCCATAACTATTTAAAT 24 N277SCTTGCTGCAATCGAAAGYTATTTAAATAAAACA 25 R310AGGCTATTTTGATATGGCAAATATTTTAAATGGT 26 E360Q TCTGACAAGGCAGCAAGGTTA 27Q391E GATCCACTTCTGGAAGCACGTCAAACG 28 W439R GGGGGTAATAAAAGAATGTATGTCGGG29 K444R ATGTATGTCGGGCGACATAAAGCTGG 30 N471D GATGGTTGGGGGGATTTCACTGTAA31 G476D TTCACTGTAAACGATGGGGCAGTTTCG 32 K484Q GGTTTGGGTGCAGCAATAAAT 33P18X TTTGAATGGCATTTGNNNAATGACGGGAACCAC Site-directed 34 A186XAAATTCACCGGAAAGNNNTGGGACTGGGAAGTA mutagenesis 35 Y243XGTGAAACATATTAAANNNAGCTATACGAGAGAT (for ΔRG/) 36 N277XCTTGCTGCAATCGAANNNTATTTAAATAAAACA M2027) 37 N471EGATGGTTGGGGGGAATTCACTGTAA 38 D128VCCAACGAATCGTTGGCAGGTAATTTCAGGTGCCTACACG Site-directed 39 G140SATTGATGCGTGGACGAGTTTCGACTTTTCAGGG mutagenesis 40 S144PTTTCGACTTTCCAGGGCGTAA (for 41 R168QGGTGTTGACTGGGATCAGCAATATCAAGAAAATCATATTTTCC K38AMY) 42 N181VCATATTTTCCGCTTTGCAAATACGGTNTGGAACAGGCGAGTG 43 E207DAATATCGACTTTAGTCATCCAGATGTACAAGATGAGTTGAAGGA 44 F272SGACGTAGGTGCTCTCGAATCTTATTTAGATGAAATGAATTGGG 45 S375PCGATAACATTCCAGCTAAAAA 46 W434R GACCTGGTGGTTCCAAGAGAATGTATGTAGGACGTCAT 47E446D AATGGCGATGGATGGGGCGATTTCTTTACGAATGGAGGATCT 48 D128XCCAACGAATCGTTGGCAGNNNATTTCAGGTGCCTACACG 49 QEYKGTTGACTGGGATGAGCGCAAACAAGAAAATCAT 50 N190F TGGATGAAGAGTTCGGTAATTATGA 51Q209 AGTCATCCAGAGGTCGTAGATGAGTTGAAGGATThe “N” in the base sequence means a mixed base of A, T, G and C, while“Y” means a mixed base of T and C.

By inserting an expression promoter region and the mutant α-amylase geneportion into the SmaI site of pKF19k again in a similar manner as theabove, the mutation-introduced gene became a template plasmid uponintroduction of another mutation. Another mutation was thus introducedin a similar manner to the above-described method.

With these mutated recombinant plasmids thus obtained as a template, PCRreaction was conducted using primers CLUBG (SEQ ID No. 9) and LADH (SEQID No. 6) or primers CLUBS (SEQ ID No. 9) and K38DH (SEQ ID No. 8) toamplify the mutated gene fragments. After they were cut with SalI, theywere inserted into the site of SalI-SmaI site of an expression vectorpHSP64, whereby various plasmids for producing mutant α-amylases wereconstructed (FIG. 1).

Example 3 Production of Mutant α-Amylases

The various plasmids for producing mutant α-amylases obtained in Example2 were each introduced into the strain Bacillus subtilis ISW1214 (leuAmetB5 hsdM1) in accordance with the protoplast method. The recombinantBacillus subtilis thus obtained was cultivated at 30+ C. for 4 days in aliquid medium (corn steep liquor, 4%; tryptose, 1%; meet extract, 1%,monopotassium phosphate, O.1%, magnesium sulfate, 0.01%, maltose, 2%,calcium chloride, 0.1%, tetracycline, 15 μg/mL). The activity of each ofthe various mutant α-amylases was measured using the supernatant of theculture medium.

Example 4 Evaluation of Amylase Productivity—1

Each of an enzyme having Pro₈₆ of ΔRG substituted with Ser (which willhereinafter be abbreviated as “P18S/ΔRG”), an enzyme having Gln₈₆substituted with Glu (“Q86E/ΔRG”), an enzyme having Glu₁₃₀ substitutedwith Val (“E130V/ΔRG”), an enzyme having Asn₁₅₄ substituted with Asp(“N154D/ΔRG”), an enzyme having Arg₁₇₁ substituted with Cys(“R171C/ΔRG”), an enzyme having Ala₁₈₆ substituted with Val(“A186V/ΔRG”), an enzyme having Glu₂₁₂ substituted with Asp(“E212D/ΔRG”), an enzyme having Val₂₂₂ substituted with Glu(“V222E/ΔRG”), an enzyme having Tyr₂₄₃ substituted with Cys(“Y243C/ΔRG”) , an enzyme having Pro₂₆₀ substituted with Glu(“P260E/ΔRG”), an enzyme having Lys₂₆₉ substituted with Gln(“K269E/ΔRG”), an enzyme having Glu₂₇₆ substituted with His(“E276H/ΔRG”), an enzyme having Asn₂₇₇ substituted with Ser(“N277S/ΔRG”), an enzyme having Arg₃₁₀ substituted with Ala(“R310A/ΔRG”), an enzyme having Glu₃₆₀ substituted with Gln(“E360Q/ΔRG”), an enzyme having Gln₃₉₁ substituted with Glu(“Q391E/ΔRG”), an enzyme having Trp₄₃₉ substituted with Arg(“W439R/ΔRG”), an enzyme having Lys₄₄₄ substituted with Arg(“K444R/ΔRG”), an enzyme having Asn₄₇₁ substituted with Asp(“N471D/ΔRG”), and an enzyme having Gly₄₇₆ substituted with Asp(“G476D/ΔRG) was assayed for amylase productivity. As a control, ΔRG wasemployed. A relative value (%) of amylase productivity was determinedfrom the amylase productivity of ΔRG set at 100%. The results are shownin Table 3. TABLE 3 Relative amylase Enzyme productivity (%) ΔRG 100P18S/ΔRG 277 Q86E/ΔRG 119 E130V/ΔRG 362 N154D/ΔRG 146 R171C/ΔRG 235A186V/ΔRG 485 E212D/ΔRG 327 V222E/ΔRG 135 Y243C/ΔRG 350 P260E/ΔRG 142K269Q/ΔRG 142 E276H/ΔRG 231 N277S/ΔRG 312 R310A/ΔRG 208 E360Q/ΔRG 162Q391E/ΔRG 127 W439R/ΔRG 312 K444R/ΔRG 112 N471D/ΔRG 292 G476D/ΔRG 296

Any one of the mutant enzymes exhibited higher amylase productivity thanΔRG, indicating that mutation heightened productivity of α-amylase inrecombinant Bacillus subtilis.

In particular, the productivity of each of E130V/ΔRG, A186V/ΔRG,E212D/ΔRG, Y243C/ΔRG, N277S/ΔRG and W439R/ΔRG was found to be at least 3times greater than that of ΔRG and above all, A186V/ΔRG exhibitedeminently high productivity of almost 5 times greater than that of ΔRG.

Example 5 Evaluation of Amylase Productivity—2

In a similar manner to the methods described in Examples 1, 2 and 3,each of an enzyme having Pro₁₈ of ΔRG/MT substituted with Thr (whichwill hereinafter be abbreviated as “P18T/ΔRG/MT”), an enzyme havingGln86 substituted with Glu (“Q86E/ΔRG/MT”), an enzyme having Glu₁₃₀substituted with Val (“E130V/ΔRG/MT”), an enzyme having Ala₁₈₆substituted with Asn (“A186N/ΔRG/MT”), an enzyme having Tyr₂₄₃substituted with Ser (“Y243S/ΔRG/MT”), an enzyme having Asn₂₇₇substituted with Phe (“N277F/ΔRG/MT), and an enzyme having Asn₄₇₁substituted with Glu (“N471E/ΔRG/MT”) was assayed for amylaseproductivity. As a control, ΔRG/MT was employed. The results are shownin Table 4. TABLE 4 Relative amylase Enzyme productivity (%) ΔRG/MT 100P18T/ΔRG/MT 200 Q86E/ΔRG/MT 144 E130V/ΔRG/MT 344 A186N/ΔRG/MT 344Y243S/ΔRG/MT 189 N277F/ΔRG/MT 256 N471E/ΔRG/MT 211

It was recognized that any one of the above-described mutant enzymesexhibited high amylase productivity compared with ΔRG/MT, and inparticular, the productivity of each of E130V/ΔRG/MT and A186N/ΔRG/MTwas at least 3 times greater than that of ΔRG/MT.

Example 6 Evaluation of Amylase Productivity—3

In accordance with the methods employed in Examples 1, 2 and 3, each ofan enzyme having Asp₁₂₈ of K38AMY substituted with Val (which willhereinafter be abbreviated as “D128V”), an enzyme having Gly₁₄₀substituted with Ser (“G140S”), an enzyme having Ser₁₄₄ substituted withPro (“S144P”), an enzyme having Arg₁₆₈ substituted with Gln (“R168Q”),an enzyme having Asn₁₈₁ substituted with Val (“N181V”), an enzyme havingGlu₂₀₇ substituted with Asp (“E207D”), an enzyme having Phe₂₇₂substituted with Ser (“F272S”), an enzyme having Ser₃₇₅ substituted withPro (“S375P”), an enzyme having Trp₄₃₄ substituted with Arg (“W434R”),and an enzyme having Glu₄₆₆ substituted with Asp (“E466D”) was assayedfor amylase productivity. As a control, K38AMY was employed. The resultsare shown in Table 5. TABLE 5 Relative amylase Enzyme productivity (%)K38AMY 100 D128V 325 G140S 209 S144P 197 R168Q 264 N181V 207 E207D 109F272S 175 S375P 115 W434R 124 E466D 212

It was recognized that compared with the wild type K38AMY, any one ofthe mutant enzymes exhibited high amylase productivity and inparticular, D128V exhibited high productivity at least 3 times greaterthan that of K38AMY.

Example 7 Evaluation of Amylase Productivity—4

A mutant enzyme S144P/N181V (which will hereinafter be abbreviated as“SPNV”) having, among the mutants shown in Example 6, S144P and N181V incombination was assayed for amylase productivity in accordance with themethod as described in Example 3. As a control, K38AMY, S144P and N181Vwere employed. The results are shown in Table 6. TABLE 6 Relativeamylase Enzyme productivity (%) K38AMY 100 S144P 197 N181V 207 SPNV 257

As a result, as shown in Table 6, a further improvement in amylaseproductivity was brought about by combined use.

Example 8 Evaluation of Amylase Productivity—5

In accordance with the methods as described in Examples 1, 2 and 3, eachof an enzyme obtained by substituting Arg₁₆₈ of the gene of aheat-resistance improved enzyme LA-K38AMY/NFQV with Gln (which willhereinafter be abbreviated as “R168Q/LA-K38AMY/NFQV”), an enzymeobtained by substituting Glu₄₆₆ of the above-described gene with Asp(“E466D/LA-K38AMY/NFQV”), and an enzyme having double mutations ofExample 6 introduced into the gene (“SPNV/LA-K38AMY/NFQV”) was assayedfor amylase productivity. As a control, LA-K38AMY/NFQV was employed. Theresults are shown in Table 7. TABLE 7 Relative amylase Enzymeproductivity (%) LA-K38AMY/NFQV 100 R168Q/LA-K38AMY/NFQV 304E466D/LA-K38AMY/NFQV 264 SPNV/LA-K38AMY/NFQV 154

As a result, it was recognized that any one of the mutant enzymesobtained in this Example exhibited high amylase productivity at leastabout 1.5 times greater than that of LA-K38AMY/NFQV and in particular,R168Q/LA-K38AMY/NFQV exhibited about 3 times greater productivity.

Example 9 Evaluation of Amylase Productivity—6

In accordance with the methods as described in Examples 1, 2 and 3, eachof an enzyme obtained by substituting Asp₁₂₈ of the gene of aheat-resistance improved enzyme LA-K38AMY/QEYK/NFQV with Val (which willhereinafter be abbreviated as “D128V/LA-K38AMY/QEYK/NFQV”) and an enzymehaving double mutations of Example 6 introduced into the gene(“SPNV/LA-K38AMY/QEYK/NFQV”) was assayed for amylase productivity. As acontrol, LA-K38AMY/QEYK/NFQV was employed. The results are shown inTable 8. TABLE 8 Relative amylase Enzyme productivity (%)LA-K38AMY/QEYK/NFQV 100 D128V/LA-K38AMY/QEYK/NFQV 602SPNV/LA-K38AMY/QEYK/NFQV 427

As a result, it was recognized that any one of the mutant enzymesobtained in this Example exhibited markedly high amylase productivitycompared with LA-K38AMY/QEYK/NFQV and in particular,D128V/LA-K38AMY/QEYK/NFQV exhibited drastic increase (about 6 times) inproductivity.

Example 10 Evaluation of Amylase Productivity—7

Into D128V/LA-K38AMY/QEYK/NFQV which was recognized to show a drasticincrease in productivity among the mutant enzymes shown in Example 9, amutation for heightening oxidant resistance by substituting Met₁₀₇ withLeu (this mutation will hereinafter be abbreviated as “M107L”) wasintroduced in accordance with the methods as described in Examples 1 and2 (“ML/DV/LA-K38AMY/QEYK/NFQV”).

Then, the gene of the mutant enzyme ML/DV/LA-K38AMY/QEYK/NFQV wasassayed for amylase productivity in accordance with the method ofExample 4. As a control, D128V/LA-K38AMY/QEYK/NFQV was employed. Theresults are shown in Table 9. TABLE 9 Relative amylase Enzymeproductivity (%) D128V/LA-K38AMY/QEYK/NFQV 100 M107L/D128V/LA- 115K38AMY/QEYK/NFQV

The relative amylase productivity of the mutant enzymeML/DV/LA-K38AMY/QEYK/NFQV was 115%, indicating that introduction ofM107L mutation for reinforcing oxidant resistance did not adverselyaffect high productivity of amylase in recombinant Bacillus subtilis.

Example 11 Evaluation of Amylase Productivity—8

In accordance with the methods as described in Examples 1, 2 and 3, anenzyme obtained by substituting Asp₁₂₈ of the gene ofheat-resistance-improved enzyme QEYK/NFQV with Gln (the resultant enzymewill hereinafter be abbreviated as “D128Q/QEYK/NFQV”) was assayed foramylase productivity. As a control, QEYK/NFQV was employed. The resultsare shown in Table 10. TABLE 10 Relative amylase Enzyme productivity (%)QEYK/NFQV 100 D128Q/QEYK/NFQV 247

It was recognized that the mutant enzyme exhibited productivity of atleast 2 times greater than that of QEYK/NFQV.

Example 12 Solubility Assay

After storage of each of the mutant enzyme preparations as shown inTable 11 at 4° C. for 1 week, the precipitate formed by centrifugation(13000 rpm, 10 minutes, 4° C.) was separated. The precipitate wassuspended in the same volume, as that before centrifugation, of aTris-HCl buffer (pH 7.0) containing of 2 MM CaCl₂. The resultingsuspension was diluted about 500-folds with the same buffer to dissolvethe former in the latter and enzymatic activity in the resultingsolution was measured. The supernatant was diluted in a similar mannerand enzymatic activity in it was also measured. Solubility of each ofthe mutant enzymes was evaluated by comparing the enzymatic activity ineach of the precipitate solution and supernatant with that of thepreparation before storage at 4° C. The results are shown collectivelyin Table 11. TABLE 11 Residual activity (%) after storage at 4° C.Enzyme Supernatant Precipitate ΔRG 55 40 ΔRG Gln86 → Glu 83 11 ΔRGPro260 → Glu 70 18 ΔRG Lys269 → Gln 74 27 ΔRG Asn471 → Asp 74 23 ΔRGLys484 → Gln 71 24

As a result, when an improved α-amylase (ΔRG) having heat resistanceimproved by deleting Arg₁₈₁ and Gly₁₈₂ was stored at 4° C. for one week,precipitation of the enzyme was recognized and only about half of theactivity remained in the supernatant. On the other hand, the mutantenzymes obtained by introducing a further mutation in ΔRG-LAMY showed ahigh activity residual ratio in the supernatant, indicating animprovement in solubility by mutation. In particular, the enzyme havingGln₈₆ substituted with Glu showed the highest enzyme solubility and 80%of the enzyme remained in the supernatant under the conditions of thisExample.

Example 13 Detergent Composition for Automatic Dish Washer

A detergent composition for automatic dish washer having the compositionas shown in Table 12 was prepared, followed by incorporation therein ofvarious mutant enzymes obtained in the productivity increasing method.As a result, the highly productive mutant enzymes exhibited similar orsuperior detergency to the control enzyme when they were equal inactivity. TABLE 12 Composition of detergent (%) Pluronic L-61 2.2 Sodiumcarbonate 24.7 Sodium bicarbonate 24.7 Sodium percarbonate 10.0 No. 1sodium silicate 12.0 Trisodium citrate 20.0 Polypropylene glycol 2.2“Silicone KST-04” (product of Toshiba Silicone) 0.1 “Sokalan CP-45”(product of BASF) 4.0Capability of Exploitation Industry

By using the mutant α-amylases according to the present invention,α-amylases are available at a high yield from recombinantmicroorganisms, making it possible to largely reduce the cost of theirindustrial production. The mutation for productivity increase in thepresent invention does not adversely affect biochemical properties ofthe enzymes so that highly productive liquefying alkaline α-amylaseshaving heat resistance, chelating agent resistance and oxidantresistance and being useful as enzymes for a detergent can be produced.

1. A mutant α-amylase which is derived from an α-amylase having an aminoacid sequence represented by SEQ ID No. 2 or showing at least 60%homology thereto by substitution or deletion of at least one amino acidresidue corresponding to any one of Pro₁₈, Gln₈₆, Glu₁₃₀, Asn₁₅₄,Arg₁₇₁, Ala₁₈₆, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, Glu₂₇₆, Asn₂₇₇,Arg₃₁₀, Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁ and Gly₄₇₆ of the aminoacid sequence.
 2. (canceled)
 3. A mutant α-amylase according to claim 1,wherein the substitution or deletion of at least one amino acid residueis substitution of the amino acid residue corresponding to Pro₁₈ withSer or Thr, the amino acid residue corresponding to Gln₈₆ with Glu, theamino acid residue corresponding to Glu₁₃₀ with Val or Gln, the aminoacid residue corresponding to Asn₁₅₄ with Asp, the amino acid residuecorresponding to Arg₁₇₁ with Cys or Gln, the amino acid residuecorresponding to Ala₁₈₆ with Val or Asn, the amino acid residuecorresponding to Glu₂₁₂ with Asp, the amino acid residue correspondingto Val₂₂₂ with Glu, the amino acid residue corresponding to Tyr₂₄₃ withCys or Ser, the amino acid residue corresponding to Pro₂₆₀ with Glu, theamino acid residue corresponding to Lys₂₆₉ with Gln, the amino acidresidue corresponding to Glu₂₇₆ with His, the amino acid residuecorresponding to Asn₂₇₇ with Ser or Phe, the amino acid residuecorresponding to Arg₃₁₀ with Ala, the amino acid residue correspondingto Glu₃₆₀ with Gln, the amino acid residue corresponding to Gln₃₉₁ withGlu, the amino acid residue corresponding to Trp₄₃₉ with Arg, the aminoacid residue corresponding to Lys₄₄₄ with Arg, the amino acid residuecorresponding to Asn₄₇₁ with Asp or Glu, or the amino acid residuecorresponding to Gly₄₇₆ with Asp.
 4. (canceled)
 5. A gene encoding amutant α-amylase as claimed in claim 1, or a vector containing saidgene.
 6. A cell transformed by a vector as claimed in claim
 5. 7. Amethod for producing a mutant α-amylase, which comprises cultivating atransformant cell as claimed in claim
 6. 8. A detergent compositioncomprising a mutant α-amylase as claimed in claim 1.